Since 1962, light transmission measurements have been utilized to detect aggregation caused by a variety of agents (Born, G. V. R., J. Physiol. 162:67P (1962)); Born, G. V. R., Nature 194:927 (1962)). In a matter of seconds after the addition of an aggregating agent it is observed a large increase in light transmission associated with the formation of aggregates. After a short but variable time, the light transmission may either decrease again indicating the dispersion of the aggregates, or increase even further. The latter is caused by an increase in the distance between the aggregates (Born G. V. R., and Hugh, M., Nature 215:1027 (1967)). The changes in transmitted light have been given a mathematical treatment based on a series of assumptions relating to the manner in which aggregation occurs (Cronberg, S., Coagulation 3:139 (1970)).
In the case of platelet aggregation, considerable evidence suggesting that adenosine diphosphate (ADP) plays an essential role (Michael, S., and Firkin, B. G., Ann Rev. Pharmacol. 9:95 (1969); Mustered, J. F., and Packham, M. A., Pharmacol. Rev. 22:97 (1970)). ADP, whether added or formed in the platelets, has been shown to cause the initial transformation in platelet morphology preceding aggregation. Ethylenediaminetetraacetate (EDTA) was added in concentrations sufficient to prevent aggregation and the plasma was then diluted to decrease the platelet concentration. It was found that the decrease in light transmission is only associated with the change and shape of the platelet and not with an increase in the main volume of the cells (Zucker, M. B., and Zaccardi, J. B., Fed. Proc. 23:299 (1964)).
The measurement of aggregation in a sample is of fundamental importance in studies of, e.g., platelet behavior. The basic technique for the measurement of aggregation utilizes changes in optical transmission of readings. Data collection was completed within 10 a suspension of aggregatable particles or material, e.g., platelets, as it is converted into a suspension of aggregates, and records these changes with a spectrophotometer fitted with, e.g., a magnetic stirrer, a thermostatted cuvette and a chart recorder. (Mustard, J. F. and Packham, M. A., Pharmacol Rev 22:97-187 (1970)).
Typically, most instruments detect the formation of aggregates by monitoring either optical turbidity or electrical conductivity. The latter represents the traditional approach employed by a so-called fibrometer-type instrument. In fact, this instrument measures increases in conductivity which may be correlated to the formation of aggregates. Similarly, turbidity may be optically sensed by the decrease in light transmission due to the formation of aggregates.
Some optical instruments permit two or four samples to be run and the results are recorded simultaneously. Other new devices allow for the electronic storage of aggregation curves. Up to the present time the applications of aggregation measurements have been restricted because of the variation between samples, the limited stability of suspensions and the cumbersome nature of individual tracings. Aggregation data are commonly reported for publication as subsequent tracings done on a single sample. These tracings are often accompanied by a footnote indicating that the data presented are representative of a number of similar experiments.
Various photometers are commercially available for measuring the light absorbance of liquid samples in microtitration plates or other sample-holding vessels. Example of such equipment are the MR 600 Microplate Reader marketed by Dynatech Laboratories, Inc. of Alexandria, Va., and the Vmax Kinetic Microplate Reader marketed by Molecular Devices of Palo Alto, Calif.
Born, G. V. R, Nature 4832:927 (1962) utilizes large volume samples (3 ml samples) contained in a centrifuge tube, the content of the tube is stirred gently with a rod and the optical density of each sample is individually measured. It is reported in this article that vigorous stirring at 1,000 rpm cycles/minute contribute to the breaking up of platelets.
Michal, F., and Born, G. V. R., Nature New Biol. 231:220 (1971) disclose a modification of the traditional optical method of measuring aggregation which permits the simultaneous measurement of scattered and transmitted light. This method encompasses a modification of the cuvette chamber of an aggregometer to allow for the measurement of light scattered at right angles to the incident light beam. In the aggregometer the incident light beams a suspension of platelets which are kept in rapid motion by a magnet rotating in the bottom of the glass tube at 1,000 rpm. The sample volume is about 1 ml and the optical density is read individually for each sample which is kept in a water-jacketed environment at 37.degree. C.
Mills, D. C. B., and Roberts, G. C. K., J. Physiol. 193:443-453 (1967) measure platelet aggregation in a modified EEL Long Cell Absorptiometer manufactured by Evans Electro-selenium Ltd., of Halstead Essex the measurements are taken of a plasma sample which is stirred from below by a magnetic stirrer while continuous recordings are made. The volume sample is about 1.0 ml. O'Brien, J. R., Nature 202:1188 (1964) conduct aggregation studies of 2 ml plasma samples placed in a cuvette in an EEL titrometer or electrophotometer. Each sample is treated individually and it is said that aggregation occurs when the optical transmission increases and is continuously recorded downwardly on the tracings.
O'Brien, J. R., J. Clin. Path. 15:446 (1962) observed the adherence of blood platelets to glass by depositing samples on a glass slide and covered it with a cover slip. The method of detection is by microscope and the viscous metamorphosis or platelet aggregation is observed. This method also utilizes large samples which are placed in glass tubes.
In recent years, microtiterplates (MP) have been increasingly used in a number of analytical applications. These disposable plastic plates contain 96 wells with a working volume of 0.3 ml per well. The wells are positioned in 12 by 8 array. Spectrophotometric devices which interface with microcomputers are commercially available which permit the rapid reading of optical changes occurring in the wells. Moreover, the thus obtained data may be electronically stored in a computer memory.
However, up to the present time there has not been available an apparatus or method for obtaining simultaneous spectrophotometric readings for a plurality of samples and controls utilizing efficient agitation, and optionally temperature conditions to follow aggregation, which could then be mathematically treated and averaged, including by computer technology, to provide statistically significant results.